Structure and function of the EA1 surface layer of Bacillus anthracis

The Gram-positive spore-forming bacterium Bacillus anthracis is the causative agent of anthrax, a deadly disease mostly affecting wildlife and livestock, as well as representing a bioterrorism threat. Its cell surface is covered by the mutually exclusive S-layers Sap and EA1, found in early and late growth phases, respectively. Here we report the nanobody-based structural characterization of EA1 and its native lattice contacts. The EA1 assembly domain consists of 6 immunoglobulin-like domains, where three calcium-binding sites structure interdomain contacts that allow monomers to adopt their assembly-competent conformation. Nanobody-induced depolymerization of EA1 S-layers results in surface defects, membrane blebbing and cell lysis under hypotonic conditions, indicating that S-layers provide additional mechanical stability to the cell wall. Taken together, we report a complete model of the EA1 S-layer and present a set of nanobodies that may have therapeutic potential against Bacillus anthracis.

• I am having a major difficulty in seeing the disruption of the S-layer in Figure 5a.This could be due to the usual problems with staining.Have the authors performed cryo-EM?If cryo-EM is not possible, could the authors please show a gallery of images for the control and treatment?o I would recommend randomising the images and asking an unbiased person to quantify and then report statistics.
• Structure of EA1 containing immunoglobulins is fascinating.Could the authors please compare the immunoglobulins to those reported from archaea (PMID: 34818541) and diderm bacteria (PMID: 37043530)?
• Line 116 -how similar is the SLH domain to previously solved structures, e.g Paenibacillus alvei?Please show alphafold's prediction for the SLH domain (PMID: 30087354).
• Line 220 -could you show a map vs model FSC for the D3-D6 fitted into the cryoEM map?How close is it to the reported resolution?
• Lines 248-249 -the mutants suggest that the X-ray structure lattice is a "true contact" but it does serve as a direct proof.Also, please explain briefly in the text what are these mutants (ΔD6 and N614W-A484Q) since this is the first time they are mentioned in the text.
• What happens when the Sap Nbs were mixed with EA1 Nbs? Cell growth should be decreased as the expression of either S-layer proteins cannot compensate for depolymerization.
• Has any Nbs mixture performed better than others at preventing EA1 polymerisation or were single Nbs and mixtures equally potent?
• Line 391 -why was the C-terminal domain truncated in the ΔD1 mutant?It is actually a ΔD1 ΔD6 mutant.Please correct this in the text.
• Line 541 -please mention that anisotropic correction was performed to obtain the X-ray crystal structure.If this is not the case, why was Staraniso used?

Minor comments
General -doi links of several papers is incorrect (Couture-Tosi, Domanska, Doran, etc), please correct these.
• Line 171 -please add a few words on SbsB for non-specialists.
• Were the authors able to identify where Nb633 binds EA1?
• Line 228 -Supplementary figure 11 mentions the cysteine mutants, however they are presented later.I would remove any mention until the relevant part of the paper.
• Line 111 -how does the predicted alphafold EA1AD compare with the experimentally determined structure?Please add a superposition of both to supplementary figure 1.
• Line 120,130 -does recombinant EA1 refer to the EA1FL or EA1AD or both?Please clarify.
• Lines 231-232 -add abbreviation to BR, BL, R in the text as well.
• Line 257 -typo, should read SC not SD.
• Line 276 -does CC refer to map vs model?Please clarify.
• Line 555 -was calcium added to the purified EA1 to facilitate polymerization?

Figures
• Figure 3 and Supplementary figure 9 -please indicate where the interacting loops with the bound calcium are present in the lattice.Do they make any connections to the neighbouring subunits?In Supplementary figure 9 it might be better to move/make transparent the 74.29 Å label to better visualize the contacts made by D6 with its surrounding subunits.
• Figure 4 -please show the MC atoms that are involved in H-bond formation in all interfaces presented.
• Supplementary figure 1 -could the authors show the alphafold prediction for Sap as well?How does the confidence score and prediction look like for Sap?
• Supplementary figure 2 -in a) please correct to "(G and S respectively as indicated on the figure)".
• Supplementary figure 4 -CDR should be abbreviated and mentioned in the text.
• Supplementary figure 7 -b) the superposition is not very informative, perhaps add transparency to each domain to visualize the similarities better.d) It would be interesting to superpose EA1 and Sap to view their overall arrangement.
• Supplementary figure 11 -how do the Cys mutants' lattices compare with the AD lattice?Perhaps adding Fourier transform of the three double mutant alongside AD would be useful.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this study, Sogues and co-authors described the structure of the surface layer protein EA1 of Bacillus anthracis.They were able to report the structure of the EA1 assembly domain, something that was assayed for years, by using nanobodies that inhibit EA1 self-assembly and depolymerize existing EA1 S-layer lattices.This is an important knowledge for the field.The manuscript is well written and well organized.

Author response:
We would like to thank Reviewer #1 for this positive assessment of our work and its contribution to the field.
My only major comment concerns the last paragraph of the result section: Effect of EA1 nanobodies on living bacteria and growth.In this paragraph, the authors observed that B. anthracis cells did not show a reduced growth in the presence of EA1 depolymerizing Nbs.They hypothesized that the loss of EA1 S-layer can be compensated by SAP expression.This can easily be tested by performing the same experiment with the ∆sap strain (strains that was used by the authors in a previous publication (Fioravanti, Mathelie-Guinlet, Dufrêne, Remaut, 2022)).It would be appreciated to include this in the manuscript.Also, what happen when EA1 Nbs are added to growing cells when they enter in stationary phase (when EA1 is already on the cell surface)?A growth curve experiment can be performed as the author did (growth in BHI in microplate) but instead of adding the nanobodies at the beginning of the growth experiment, Nbs can be added at the entry of stationary growth phase.
Author response: As suggested by Reviewer #1, we now more elaborately investigate the possible biological activity of EA1 depolymerising Nbs.The growth curve shown in Figure 5 of the original manuscript was done using an overnight, stationary phase culture as inoculum, and therefore representing an inoculum with maximum presence of EA1 when starting the growth curve.However, when resuming growth, the EA1 S-layer is expected to be readily replaced by Sap.We now include the suggested experiment using the ∆sap strain, but find no growth delay or defects when using EA1 nanobodies (Figure 5c), thus ruling out the hypothesis that a lack of phenotype from the EA1 nanobodies stems from a compensatory expression of the Sap Slayer.In the Fioravanti et al. 2022 study we identified the Sap S-layer as a mechanical support to the cell envelope, and showed that acute loss of a crystalline Sap S-layer by Nb treatment resulted in cell lysis during hypo-osmotic stress.Therefore, we decided to test the activity of the EA1 nanobodies by exposing the treated cells to an osmotic downshift prior to inoculation in BHI medium.This showed that when exposed to hypotonic conditions, the WT strain fully lost viability when treated with nanobody Nb643 (Figure 5.d).When inspected by microscopy, these cells showed membrane blebbing and cell lysis, indicating that in absence of a crystalline EA1 S-layer the cell envelope has a lowered ability to provide mechanical resistance to the cell turgor.Remarkably, when cells of the ∆sap strain were treated with Nb643 and exposed to an osmotic downshift, growth curves only showed a partial loss in viability in the form of an extended lag phase (Figure 5.e).Repeated treatment cycles showed a reproducible delay in lag phase, and microscopic inspection showed the ∆sap cells were less prone to osmotic lysis upon loss of the EA1 S-layer, thus suggesting the presence of an osmoadaptive mechanism in the mutant lacking Sap.These new results are in line with observations made in Fioravanti, Mathelie-Guinlet, Dufrêne, Remaut, 2022, and show that an acute loss of a crystalline S-layer results in a cell envelope with reduced mechanical resistance to low osmolality conditions.An effect that can be readily compensated in cells that chronically lack an S-layer, however, by a hitherto unknown mechanism.
Minor correction: Line 85 « (SLH) that anchor the protein to the cell surface through binding of the ketal pyruvylated N-acetylmannosamine unit in the peptidoglycan via non-covalent interactions" Please replace peptidoglycan by Secondary Cell Wall Polysaccharide (SCWP) End of line 147, a bracket is missing L194 and l196, please replace fig 2g by fig 2f Line 293, replace levers by levels Line 327, correct EA by EA1 Line 338, correct heteropmorphic by heteromorphic Line 600, a word is missing after 20, is it 20 min?